Bone integrity is maintained via the careful balance of bone deposition by osteoblasts and bone resorption by osteoclasts (OC). Bone remodeling occurs throughout life and imbalances in formation and resorption lead to human diseases including osteoporosis and periodontitis, a mission of the agency. We are studying the negative regulation of ITAM-adapter signals in osteoclasts. Specifically we are investigating the possibility that phosphatases may regulate ITAM-adapter, DAP12, signaling. We have found a novel association of activated DAP12 with SH2-containing inositol-5'-phosphatase 1 (SHIP1). Our central hypothesis is that SHIP1 regulates DAP12 signaling during OC development and function in vitro and in vivo. Specific Aim 1: Determine the mechanism by which SHIP1 inhibits DAP12 signaling. These studies aim to determine the functional domains of SHIP1 that are required for inhibition of DAP12 signaling, the role of proximal kinases, and the specific downstream signaling pathways negatively regulated by SHIP1. We will determine the role of SHIP1 in negatively regulating DAP12 downstream of MCSF, RANKL, and integrin stimulation. We will investigate the specific role of DAP12-associated receptors in mediating SHIP1- DAP12 association. Specific Aim 2: Determine how SHIP1 inhibition of DAP12 signaling affects the OC functions of resorption, actin ring formation and survival in vitro. We will determine the cellular localization of SHIP1 and DAP12 during DAP12 stimulation. Specific Aim 3: Determine the affect of SHIP1 on DAP12 signaling in vivo. We will determine whether SHIP1 regulates DAP12 signaling in vivo after direct activation of DAP12 with anti-TREM2 antibodies or blockade of DAP12 with TREM2-fusion protein. Specific Aim 4: Determine the role of SHIP1 and TREM2/DAP12 in response to Porphyromonas gingivalis (P. gingivalis) induced alveolar bone loss. Additionally we will investigate the roles of DAP12 and SHIP1 in inflammatory trabecular and alveolar bone loss induced by chronic low dose LPS treatment or acute stimulation with P. gingivalis LPS in vivo. These studies will not only elucidate how SHIP1 regulates DAP12 signaling in OC but will potentially give insights into how DAP12 can function as both an activating and inhibitory signal in macrophages and OC. This understanding might lead to novel therapeutic interventions in periodontal disease and osteoporosis. Project Narrative: The aim of proposed studies is to define a key regulatory pathway needed to inhibit activity of osteoclasts, cells that dissolve bone. This understanding might lead to novel therapeutic interventions in chronic periodontal disease and osteoporosis, diseases associated with excessive osteoclast activity.